Viridibacillus soli sp. nov., isolated from forest soil in Ailaoshan National Nature Reserve

A Gram stain-positive, rod-shaped, and subterminal endospore-forming bacterium, designated strain YIM B01967T, was isolated from a forest soil sample collected in Ailaoshan National Nature Reserve, Yuxi City, Xinpin county, Yunnan province, China. Strain YIM B01967T showed the highest 16S rRNA gene sequence similarity with Viridibacillus arvi (99.1%) and Viridibacillus arenosi (98.9%). Based on the phylogenetic and 16S rRNA gene sequence results, strain YIM B01967T was affiliated to the genus Viridibacillus. The growth of YIM B01967T was observed at 15–35 °C (optimum, 28 °C), pH 7.0–9.0 (optimum, pH 7.5) and in the presence of 0–2% (w/v) NaCl (optimum in 2% NaCl). The cell wall sugars include ribose, glucose, arabinose, galactose, and mannose. The quinone system consisted of the major compound MK-8 and moderate amounts of MK-7. The major fatty acids (> 10%) included iso-C15:0, anteiso-C15:0, C16:1ω10c. The major polar lipids profile included DPG, PME. The cell wall peptidoglycan was most likely of the type A4α with an l-Lys-d-Asp interpeptide bridge. The genomic DNA G + C content of strain YIM B01967T was 36.3 mol%. The ANI and digital DNA–DNA hybridization (dDDH) values between strain YIM B01967T and Viridibacillus arvi DSM 16317 T, Viridibacillus arenosi DSM 16319 T were 61.0% and 32.1%, 60.0% and 33.1% based on the draft genome sequence. The results support the conclusion that strain YIM B01967T represents a novel species of the genus Viridibacillus, for which the name Viridibacillus soli sp. nov., is proposed. The type strain is YIM B01967T (= KCTC 43249 T = CGMCC 1.18436 T).


Introduction
The genus Viridibacillus belongs to the family Planococcaceae, phylum Bacillales which was first proposed by Albert et al. (2007) to reclassify some species in the genus Bacillus. Up to now, this genus consists of 3 species, namely Viridibacillus arenosi (Heyrman et al. 2005;Albert et al.2007), Viridibacillus arvi (Heyrman et al. 2005;Albert et al.2007), and Viridibacillus neidei (Nakamura et al. 2002;Albert et al.2007), all isolated from soil. In this study, to study the soil microbial species diversity in the extreme environment of the alpine mountainous area, the strain YIM B01967 T was isolated from a forest soil sample in Ailaoshan National Nature Reserve, Yuxi City, Xinpin county, Yunnan province, China. Through the study of polyphasic taxonomy,

Strain isolation and culture conditions
Forest soil surface samples were collected from the Ailao Mountain National Nature Reserve in Yuxi City, Yunnan Province, China. Isolation was performed by the standard dilution plate method on plate count agar (PCA; Difco) at 28 °C for 4-7 days. The isolation procedure was performed as described by Liu et al. (2017). The pure culture of the randomly selected single colony strain YIM B01967 T was stored on a PCA slant at 4 °C, and stored as a glycerol suspension (20%, w/v) at − 80 °C. Besides, it was preserved in lyophilized form in skimmed milk at 4 °C temperature.
The reference strain, Viridibacillus arvi DSM 16317 T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).

Phylogenetic and genotypic analyses
The preliminary identification of strain YIM B01967 T was performed based on the 16S rRNA gene sequence and phylogenetic analysis. The extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Li et al. (2007). The closest phylogenetic neighbours and the corresponding similarity were determined by aligning the obtained 16S rRNA sequence against the bacterial type species recorded in the EzBio-Cloud server (https: //www. ezbio cloud. net; Yoon et al. 2017). Multiple sequence alignments were performed with CLUSTAL_X (Thompson et al. 1997). Phylogenetic analysis was performed using the MEGA software package version 7.0 (Kumar et al. 2016). The phylogenetic trees were reconstructed using neighbor-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) (Fitch 1971) with MEGA 7 software (Kumar et al. 2016). The method used to compute evolutionary distances was Kimura's two-parameter (Kimura 1980). The stability of the topology and the phylogenetic tree was evaluated using bootstrap analysis (Felsenstein 1985), with 1000 replications. The 16S rRNA gene sequence of Bhargavaea cecembensis DSM 22132 T was used as an outgroup. The sequencing of the whole genome was performed on the HiSeq X-Ten platform (Illumina). The draft wholegenome sequencing of the strain YIM B01967 T was performed by Majorbio (Shanghai, China). Average Nucleotide Identity (ANI) values were calculated using JSpeciesWS (http:// jspec ies. riboh ost. com/ jspec iesws/# analy se) (Richter et al. 2016). The Genome-to-Genome Distance Calculator, version 2.1 was used to calculate the digital DNA-DNA hybridization (dDDH) value (Meier-Kolthoff et al. 2013). The dDDH results of recommended formula 2 (identities/ HSP length) were used. A phylogenomic tree was constructed based on genomic data using the supermatrix method (Zhi et al. 2017).

Morphological, physiological, and biochemical analyses
To determine the differential phenotypic properties, strain YIM B01967 T was subjected to morphological, physiological, and biochemical analyses. Phenotypic characteristics of strain YIM B01967 T were observed using cells grown on PCA medium for 4 days at 28 °C. Cell morphology was examined using a transmission electron microscope (JEM 2100; JEOL). For transmission electron microscopy, cells were negatively stained with 1% phosphotungstic acid before observation. Colony morphology and pigmentation were observed on PCA medium incubated at 28 °C for 4 days. Growth at different temperatures (4, 10, 15, 20, 25, 28, 30, 35, 37, 40, and 45 °C) was examined after incubation on PCA medium for 4 days. Tolerance to NaCl between 0 and 10% (w/v, at intervals of 2%) in plate count broth (PCB; Difco) medium at 28 °C was recorded after 4 days. The ability of the strain to grow at different pH values (4.0-10.0, at 0.5 intervals using the buffer system described by Tang et al. 2010). Catalase activity was determined by the production of bubbles after adding 3% H 2 O 2 to the tested bacteria (Tarrand and Gröschel 1982). Enzyme activities, production of acid, utilization of different compounds, and the other physiological functions were tested with API ZYM, API 20NE kits (bioMérieux), API 50CHB kits, and the Biolog GEN III MicroPlates kits according to the manufacturers' instructions. All tests were completed in duplicate.

Chemotaxonomic characterization
The isolation of the peptidoglycan and analysis of the peptidoglycan structure were done according to published protocols (Schumann 2011;Schleifer and Kandler 1972). Analyses of cell wall peptidoglycan and sugars of whole-cell hydrolysates were performed according to the procedures described by Lechevalier and Lechevalier (1970) and Tang et al. (2009). Cellular fatty acids were extracted, methylated, and analyzed using the Sherlock Microbial Identification System (MIDI) according to the manufacturer's instructions. Fatty acid methyl esters were analyzed using the Microbial Identification Software Package (Sherlock Version 6.1; MIDI databaseTSBA6) (Sasser 1990). The respiratory quinones of YIM B01967 T were extracted from lyophilized cells (Collins and Jones, 1980), purified by TLC, and then analyzed by HPLC according to the methods of Xie and Yokota (2003). Polar lipids were extracted, examined by two-dimensional TLC, and identified using the procedures described by Collins and Jones (1980) and Minnikin et al. (1979).

Molecular phylogenetic analysis
The almost-complete 16S rRNA gene sequence of YIM B01967 T was 1540 bp (GenBank accession number MW386301). Strain YIM B01967 T showed the highest 16S rRNA gene sequence similarity with Viridibacillus arvi (99.05%) and Viridibacillus arenosi (98.92%). The NJ tree, MP tree, and ML tree for the 16S rRNA shared the same topology and are presented in Fig. 1, Fig S1, and Fig S2, respectively.
The draft genome of strain YIM B01967 T contained 112 contigs, with a total length of 4,553,251 bp and an N50 length of 171,059 bp (GenBank accession number JAEOAH000000000), and genome coverage of 14.0 × . The DNA G + C content of strain YIM B01967 T was determined from the genome to be 36.3 mol%. Strain YIM B01967 T genome was annotated with 4444 genes, included 4200 protein-coding genes, 60 rRNA genes, 50 tRNA genes, 5 ncRNA genes, and 184 pseudogenes. In contrast, the draft genome of the reference strain Viridibacillus arvi DSM 16317 T consists of 4,758,570 bp with an N50 contig length of 244,670 bp and a G + C content of 35.0 mol%. The ANI values between strain YIM B01967 T and its closely related strains, Viridibacillus arvi DSM 16317 T and Viridibacillus arenosi DSM 16319 T , were 61.0% and 60.0%, respectively, based on the draft genome sequence, which was lower than the 95.0% cut-off for species demarcation (Wayne et al. 1987;Richter et al. 2016). The dDDH values between strain YIM B01967 T and Viridibacillus arvi DSM 16317 T , Viridibacillus arenosi DSM 16319 T were 32.1% and 33.1% based on the draft genome sequence, which was much lower than the threshold value (70%) recommended for distinguishing novel prokaryotic species (Goris et al. 2007;Chun et al. 2018). The phylogenomics tree of YIM B01967 T with the closely related strains is presented in Fig. 2.

Morphological, physiological, and biochemical analyses
The YIM B01967 T strain was Gram positive, sporulating and active rod shaped. The endospores were approximately round and are located in the sporangia with enlarged or slightly enlarged ends. (Fig S1). Cells can grow at in the presence of 0-2% (w/v) NaCl (optimum in 2% NaCl). Other physiological characteristics are given in Table 1. Strain YIM B01967 T was catalase-positive and oxidase-negative, and this feature was also present in Viridibacillus arvi DSM 16317 T and Viridibacillus arenosi DSM 16319 T .
The detailed differentiating phenotypic and chemotaxonomic characteristics features between YIM B01967 T and the reference strains are given in Table 1.
Consequently, based on the above findings, we characterized strain YIM B01967 T as a novel species within the genus Viridibacillus, for which the name Viridibacillus soil sp.nov.is proposed.
The type strain, YIM B01967 T (= KCTC 43,249 T = CGMCC 1.18436 T), was isolated from a soil sample collected from a forest in Ailaoshan National Nature Reserve, Yuxi City, Xinpin county, Yunnan province, China.  The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence and the draft genome sequence are MW386301 and JAEOAH000000000, respectively.